Neuroblastoma (NB) accounts for 15% of all childhood cancer deaths. Survival for children with high-risk disease remains below 40% and survival curves have plateaued despite treatment intensification strategies. As progress has stagnated, novel treatment approaches are clearly needed. Our laboratory has recently discovered that in NB two processes central to tumor progression are regulated by S1P signaling and can be targeted for therapeutic benefit. The first process concerns cell fate. Inside cells, the delicate balance between pro-survival (S1P) and apoptosis (sphingosine, ceramide) signaling is dictated by sphingosine kinases (SphK1 and SphK2). Neuroblastoma is unique among solid tumors we have evaluated in its disproportionate reliance on SphK2, which we strategically exploit, weighting the pendulum towards cell death. We have accomplished this by utilizing the sphingosine analogue FTY720, which is avidly and specifically phosphorylated by SphK2, thereby acting as a competitive inhibitor increasing pro-apoptotic sphingosine levels, while diminishing S1P levels. Secondly, we have found that extracellular S1P modulates tumor-stromal interactions. The S1P receptor, S1P2, contributes to the regulation of the tumor microenvironment in NB through expression of the potent macrophage chemoattractant MCP-1/CCL2, as well as, angiogenic factor VEGF. As we show, selective interference with this process disrupts establishment of a milieu favorable for tumor progression. Using this novel 2-pronged approach we demonstrate efficacy and synergy with current chemotherapy. Thus, our overarching hypothesis is that rational exploitation/inhibition of S1P signaling mechanisms will synergize with cytotoxic agents and result in enhanced treatment efficacy. This hypothesis is explored in 3 aims in this proposal. Aim 1: Evaluates the efficacy of FTY720 based NB tumor inhibition- In this aim we explore the hypothesis that FTY720 based interference with over-expressed SphK2 in NB cells results in enhanced apoptotic signaling through caspase independent pathways, favoring tumor cell death. These studies will evaluate FTY720 activity, alone and in combination with topoisomerse inhibitors across selected NB cell lines, xenografts and in the MYCN transgenic model. The mechanisms involved in FTY720 related cell death will also be defined as this information may aid in future drug development in aim 3. Aim 2: Utility of S1P2 interference/inhibition strategies for treating neuroblastoma-Through induction of MCP-1/CLL2 and VEGF, S1P2 regulates macrophage invasion and angiogenesis, respectively. We will test the therapeutic effect of S1P2 receptor inhibition alone (JTE-013) and in combination therapy against NB cell lines, xenografts and the MYCN transgenic model. To determine if S1P2 is critical to tumor development and progression we will use shRNA to knock down S1P2 in NB cell lines and xenograft models. Further, we will cross transgenic MYCN mice with S1p2-/- mice available in our lab, assessing the significance of S1P2 using a genetic model. Aim 3: Development and testing of FTY720 and JTE013 analogs with improved anti-cancer properties- The first generation drugs used in aims 1 and 2 have limitations and can be associated with off-target effects. In this final aim we will test analogs of FTY720, which are designed not to bind to the S1P receptors, to reduce immunosuppressive properties and other off target effects, yet are able to target intracellular SphK2 inhibition and thereby achieve tumor cytotoxicity. In the case of JTE-013, longer in-vivo half-life and enhanced inhibitory activity are focuses for 2nd generation compounds, several compounds designed with these endpoints are evaluated using our established models. The final aim will be performed in collaboration with Dr. Rolf Swenson of Arroyo Biosciences.